Sexually Dimorphic Alterations in the Transcriptome and Behavior with Loss of Histone Demethylase KDM5C

Chromatin dysregulation has emerged as a major hallmark of neurodevelopmental disorders such as intellectual disability (ID) and autism spectrum disorders (ASD). The prevalence of ID and ASD is higher in males compared to females, with unknown mechanisms. Intellectual developmental disorder, X-linked syndromic, Claes-Jensen type (MRXSCJ), is caused by loss-of-function mutations of lysine demethylase 5C (KDM5C), a histone H3K4 demethylase gene. KDM5C escapes X-inactivation, thereby presenting at a higher level in females. Initially, MRXSCJ was exclusively reported in males, while it is increasingly evident that females with heterozygous KDM5C mutations can show cognitive deficits. The mouse model of MRXSCJ, male Kdm5c-hemizygous knockout animals, recapitulates key features of human male patients. However, the behavioral and molecular traits of Kdm5c-heterozygous female mice remain incompletely characterized. Here, we report that gene expression and behavioral abnormalities are readily detectable in Kdm5c-heterozygous female mice, demonstrating the requirement for a higher KDM5C dose in females. Furthermore, we found both shared and sex-specific consequences of a reduced KDM5C dose in social behavior, gene expression, and genetic interaction with the counteracting enzyme KMT2A. These observations provide an essential insight into the sex-biased manifestation of neurodevelopmental disorders and sex chromosome evolution.


Introduction
Mutations in methyl histone regulators are overrepresented in neurodevelopmental disorders (NDDs) such as intellectual disabilities and autism spectrum disorders [1][2][3][4][5]. The prevalence of these conditions is higher in males than in females: approximately 1.5-fold for intellectual disability and 4-fold higher for autism spectrum disorders [6,7]. Biological differences such as sex chromosomes and hormones may contribute to sex-biased prevalence. However, the diagnosis of these conditions is based on the assumption that behavioral manifestations of a specific brain malfunction are the same between the human sexes. The validity of the assumption is questionable given the above biological differences, and the incorrect assumption could contribute to the sex-biased prevalence [8]. Thus, understanding the sex-specific impact of methyl histone dysregulation is necessary to grasp the prevalence and manifestation of NDDs accurately.

Mice
Generation of the Kdm5c-KO allele was previously described by Cre-mediated deletion of exons 11 and 12 [14]. For sequencing experiments, the Kdm5c-KO allele was maintained on a mixed background of C57BL/6J and 129S1/SvImJ. Samples for sequencing were collected from 3 litters. Sex was determined through genotyping primers for Uba1 on the X and Y chromosomes with the following primers: 5 -TGGATGGTGTGGCCAATG-3 , 5 -CACCTGCACGTTGCCCTT-3 . Deletion of Kdm5c was determined through the primers 5 -ATGCCCATATTAAGAGTCCCTG-3 , 5 -TCTGCCTTGATGGGACTGTT-3 , and 5 -GGTTC TCAACACTCACATAGTG-3 . For behavioral experiments, F1 hybrids were generated by crossing female mice carrying the Kdm5c-KO allele on a congenic C57BL/6J background with mice heterozygous for loss of Kmt2a (Kmt2a-HET) on a congenic 129S1/SvImJ background, as previously described [13]. All mouse studies complied with the protocols (PRO00010350: Iwase and PRO00008807: Tronson) by the Institutional Animal Care & Use Committee (IACUC) of The University of Michigan.

mRNA-Seq
The forebrain (hippocampus and cortex) was micro-dissected from postnatal day six (P6) mice. We used four animals per genotype. Total RNA was purified and DNAse-treated by the Qiagen RNEasy Midi Kit (Qiagen #75144) after homogenizing the samples in Buffer RLT with an electric homogenizer. Purified RNA was sent to Novogene for sample quality control and cDNA library preparation by poly A enrichment. Libraries were prepared using the NEBNext ® Ultra™ II Directional RNA Library Prep Kit with oligo-dT priming and sequenced through the Illumina NovaSeq 6000 sequencing platform to generate pairedend 150 base-pair reads. After ensuring read quality via FastQC (v0.11.8), reads were then mapped to the mm10 Mus musculus genome (Gencode) using STAR (v2.5.3a), during which we removed duplicates and kept only uniquely mapped reads. Count files were generated by FeatureCounts (Subread v1.5.0), and BAM files were converted to bigwigs using deeptools (v3. 1.3) and visualized by the UCSC genome browser. RStudio (v3.6.0) was then used to analyze counts files by DESeq2 (v1. 26.0) [34] to identify differentially expressed genes (DEGs) with a q-value (p-adjusted via FDR/Benjamini-Hochberg correction) less than 0.1. The DEseq2 design included a grouping variable to test the interaction between sex and genotype, and the log2 fold changes were calculated with lfcShrink using the default apeglm package [35]. We did not perform batch-effect correction because RNA isolation, library preparation, and sequencing were performed in a single batch. MA-plots were generated by ggpubr (v0.4.0), and Eulerr diagrams were generated by eulerr (v6.1.1). Boxplots and scatterplots were generated by ggplot2 (v3.3.2). Gene ontology (GO) analyses were performed by Metascape [36] at http://metascape.org (accessed on 31 March 2022). We performed GO analysis on upregulated and downregulated genes separately, as this approach improves the identification of relevant ontologies [37]. The codes used in this study are available at https://github.com/umich-iwase-lab/2022_Kdm5cMalevsFemale (accessed on 31 March 2022). Raw sequencing data is deposited at Gene Expression Omnibus, GSE206346 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE206346 (accessed on 31 March 2022).

Behavioral Paradigms
Eighty-one adult female mice (21 wild type (WT), 13 Kdm5c-HET, 29 Kmt2a-HET, and 16 double mutant (DM)), at least 4 months old at the beginning of experiments, underwent behavioral testing. Prior to behavioral testing, mice were acclimated to the animal colony room for at least one week, singly housed in standard cages, and provided with a standard lab diet and water ad libitum. A 12-h light-dark cycle (7:00 a.m.-7:00 p.m.) was maintained at 20 ± 2 • C. All testing was conducted by experimenters masked to genotype. Mice were tested in batches as they became available, with no differences in performance across batches. All mice were tested in all behavioral tasks. To avoid confounds between tests, stressful tasks (context fear conditioning and resident intruder tests) were performed last.

Contextual Fear Conditioning
Foreground context fear conditioning was assessed as previously described [13,38]. Mice were placed into a distinct context with white walls (9 3 4 × 12 3 4 × 9 3 4 in) and a 36-steel-rod grid floor (1/8 in diameter; 1 4 spaced apart) (Med-Associates, St. Albans, VT, USA) and allowed to explore for 3 min, followed by a 2-s 0.8 mA shock, after which mice were immediately returned to their home cages in the colony room. Twenty-four hours later, mice were returned to the context, and freezing behavior was assessed with an NIR camera (VID-CAM-MONO-2A) and VideoFreeze (MedAssociates, St Albans, VT, USA). Data were analyzed using a one-way, between-groups ANOVA with genotype as the between-subjects factor, and Bonferroni corrections for post-hoc tests were used to correct for multiple comparisons.

Three-Chambered Social Interaction
Mice were placed into a three-chambered apparatus consisting of one central chamber (24 × 20 × 30 cm 3 ) and two identical side chambers (24.5 × 20 × 30 cm 3 ), each with a mesh enclosure (8 cm diameter; 18 cm height; grey stainless-steel grid 3 mm diameter spaced 7.4 mm apart). All mice were habituated to the apparatus without other mice for 10 min, 24 h prior to the test. In the interaction test, a 2-to 3-month-old stranger female mouse (C57BL/6N) was placed in the mesh enclosure on one side of the box ("stranger"), and a toy mouse approximately the same size and color as the stranger mouse on the other ("toy"). Exploration of either the stranger or toy was defined as nose-point (sniffing) within 2 cm of the enclosure and used as a measure of social interaction [13]. Animals that did not interact with the stranger mouse at all were excluded from the analyses (n = 2). Behavior was automatically scored by Ethovision XT9 software as described above, and social preference was defined as time exploring strangers/total exploration time. Social preference was analyzed using one-sample t-tests for each genotype, and groups were compared using a one-way ANOVA, with Bonferroni-corrected post-hoc tests being used to correct for multiple comparisons across genotypes.

Social Dominance Tube Test
Mice were habituated to a clear plastic cylindrical tube (1.5 in diameter; 50 cm length) and allowed to explore and enter the tube for 10 min, 24 h prior to testing. The test was conducted by placing two mice of different genotypes at opposite ends of the tube and allowing them to walk to the middle. The trial was terminated when one mouse (submissive mouse) retreated until it exited the tube with all four paws. This was scored as a loss for the submissive mouse and a win for the mouse that remained in the tube (the dominant mouse). Each mouse was tested against three different opponents, each of a different genotype, counterbalanced across groups. Videos were recorded by Ethovision XT9 software as described above, and videos were manually scored by trained experimenters with genotypes masked. The number of "wins" was reported as a percentage of the total number of matches. Data were analyzed using an Exact Binomial Test with 0.5 as the probability of success (win or loss).

Resident-Intruder Test
To test whether Kdm5c-HET, Kmt2a-HET, or DM females exhibited aggression by fighting, a behavior that is unusual in virgin mus musculus females, we conducted the resident intruder test as previously described [13]. We observed no fighting by any mouse of any genotype (data not shown).

Kdm5c-Heterozygous Female Brains Show Apparent Gene Expression Changes Similar to Male Mutants
To assess the role of lysine demethylase 5C (KDM5C) in neurodevelopment across sexes, we performed bulk mRNA sequencing (mRNA-seq) of postnatal day 6 (P6) forebrains, encompassing the cortices and hippocampi. The genotypes of animals were wildtype (WT, both male and female), Kdm5c hemizygous knockout male (Kdm5c-KO), and Kdm5c heterozygous female (Kdm5c-HET) mice. We have previously demonstrated that targeted deletion of mouse exons 11 and 12 abolishes KDM5C's enzymatic function and protein production [14]. We first confirmed the absence of mRNA-seq reads of exons 11 and 12 in Kdm5c-KO males and approximately half the expression in Kdm5c-HET females compared to their sex-matched WT controls ( Figure 1A). We next assessed Kdm5c expression in WT males and females, as KDM5C is known to escape X-inactivation in multiple species and at varying degrees across tissues [39][40][41]. Indeed, we found the Kdm5c expression in female forebrains to be approximately 1.5-fold higher than in male brains (Welch's t-test, p = 1.15 × 10 −5 ) ( Figure 1B). Principal component analysis (PCA) demonstrated that sex rather than Kdm5c genotypes accounts for transcriptome variability overall ( Figure 1C).
We next evaluated the impact of Kdm5c loss on the male and female transcriptome by identifying differentially expressed genes (DEGs) with DESeq2 (q < 0.1). Kdm5c-KO males showed 708 genes upregulated and 371 genes downregulated compared to WT males. Notably, gene expression changes were also evident in Kdm5c-HET females, albeit with lower DEG numbers, 122 up and 14 down, compared to males ( Figure 1D,E). Most DEGs are upregulated in the mutants, consistent with KDM5C being a transcriptional repressor [14]. In our previous RNA-seq study of the adult (4-8 months) male Kdm5c-KO hippocampus with identical experimental procedures, mutation, and analytical pipeline, 271 genes were upregulated, and 73 genes were downregulated [13]. Thus, the total DEG number of the male Kdm5c-KO P6 forebrain was >3-fold larger than the adult hippocampi, suggesting a pronounced vulnerability of neuronal maturation processes to Kdm5c loss. While most upregulated genes were shared between Kdm5c-HET females and Kdm5c-KO males, some genes showed significant changes only in one sex ( Figure 1F). To assess the degree of dysregulation between sexes, we then plotted the log2 fold change (Log2FC) of shared and uniquely upregulated DEGs in Kdm5c-KO males versus Kdm5c-HET females. We found that the DEGs with the greatest Log2FC from WT were those dysregulated in both sexes ( Figure 1G, left), and females exhibited 40% of the extent of male dysregulation of these shared genes ( Figure 1G, right).
These results indicate the following. First, KDM5C heterozygosity is sufficient to cause readily detectable gene expression changes in females. Second, genes relevant to known manifestations of KDM5C deficiency are commonly misregulated between male and female mutants. Third, the degree of dysregulation of the common genes was milder in Kdm5c-HET females than in Kdm5c-KO males. The last point provides insight into sex chromosome evolution (See Section 4).

Kdm5c Deficiency Also Leads to Sex-Specific Gene Expression Changes
In addition to the common gene expression changes between the sexes, we also found 847 male-specific DEGs and 20 female-specific DEGs (q < 0.1, Figure 1F,G). To gain insights into the sexually dimorphic consequences of KDM5C deficiency, we performed gene ontology (GO) analysis on DEGs upregulated in Kdm5c-KO males or Kdm5c-HET females (708 and 122 genes, respectively) with Metascape [36]. Many ontologies related to cell projection and adhesion were significantly enriched in Kdm5c-KO males (Figure 2A). Contrastingly, up DEGs for Kdm5c-HET females are enriched for ontologies relating to cellular development ( Figure 2B, such as "regulation of neural precursor cell proliferation" (GO:2000177, p = 0.0004). While there were not enough downregulated DEGs in Kdm5c-HET females (n = 14) to perform ontology analysis, Kdm5c-KO male downregulated DEGs had significant ontologies that positively regulate synapse formation and function ( Figure 2A)consistent with our previous findings that male Kdm5c-KOs have decreased dendritic spine density in the hippocampus and amygdala [13].
In females, key development-related genes contributed to the female-specific ontology enrichment. One gene of interest is midkine (Mdk), with a slight but significant upregulation in Kdm5c-HET females but not in Kdm5c-KO males ( Figure 2D, Table 1). Mdk is a neurotrophic factor expressed transiently during embryogenesis to promote neurite outgrowth [51,52]. Another female-specific DEG, JPX transcript, XIST activator (Jpx) is a long-noncoding RNA (lncRNA) crucial for X-inactivation, a females-specific epigenetic process [53,54] (Figure 2D, Table 1). Jpx is upregulated as both male and female embryonic stem cells differentiate, but the gene promotes X-inactivation only in females by inducing Xist expression [53,54]. All differentially expressed up-and downregulated genes found in males and females are listed in Supplementary Table S1.
Altogether, our transcriptomic data demonstrate that loss of Kdm5c in males and females has a sexually dimorphic impact on a subset of genes. Upregulation of early developmental genes represents the female mutant, while aggression and memory-related genes represent the male mutant.

Kdm5c and Double Kdm5c and Kmt2a Mutations Result in Social Behavior Deficits in Females
In the 3-chambered social interaction task ( Figure 3D,E), both WT and Kmt2a-HET females showed a significant preference for interaction with the stranger mouse over the toy mouse (one-sample t-test WT: p = 0.046, Kmt2a: p = 0.012), DMs showed no preference (p = 0.068), and Kdm5c-HETs showed a significant avoidance of the mouse and preference for the toy (p < 0.001). This pattern was also evident in comparing between genotypes, where a significant overall effect of genotype (F(3,78) = 8.053, p < 0.001, η p 2 = 0.244) was driven by a significant decrease in social preference by Kdm5c-HET females compared with both WT (p = 0.001) and Kmt2a (p < 0.001). DMs, despite showing no social preference and a trend towards avoidance, were not different from any other genotype, albeit with a trend towards decreased preference compared to Kmt2a mice (DM vs. WT: p = 0.125; vs. Kmt2a: p = 0.057; vs. Kdm5c p = 0.497) Figure 3D). Importantly, this is not driven by total interaction time. Across genotypes, animals interacted with stranger or toy a similar amount of time (time stranger + time toy; F(3,78) < 1) ( Figure 3E). This pattern suggests that both Kdm5c-HET and DM females are impaired in social preference but that Kmt2a knockdown in Kdm5c-HETs nevertheless may decrease avoidance of the stranger mouse. A different pattern emerged in the social dominance tube test; DM but not Kdm5c-HET female mice showed a submissive phenotype compared with WT mice (p < 0.001), with no differences between WT, Kmt2a-HET, Kdm5c-HET female mice (all p = 1) ( Figure 3F). This result was in contrast to patterns observed in males where, as previously described, both Kmt2a-HET and Kdm5c-KO males showed increased dominance over WT, whereas DM males, like the female DMs, showed a submissive phenotype [13].
Together, these results demonstrate that Kdm5c-HET females show deficits in specific aspects of social behaviors, most notably avoidance of other animals without a change in social dominance. Although Kmt2a-HET mice showed no overt changes in either social preference or dominance behaviors, the female double Kdm5c-HET, Kmt2a-HET mutants showed modifications, reducing avoidance and-similar to the male DM mice-driving a striking increase in submissive behaviors.

Double Kmt2a and Kdm5c Mutations Mediate Context Fear Memory Impairment in Females
We next used behavioral tasks to examine the impact of Kdm5c deficiency and its restoration by Kmt2a heterozygous deletion on context fear memory and social behaviors in female mice. To this end, we compared WT, Kdm5c-HET, Kmt2a-HET, and double mutants (Kdm5c-HET, Kmt2a-HET, DM) on foreground context fear conditioning ( Figure 3A-C). We observed a significant main effect of genotype on Context Fear Conditioning (F(3,81) =5.08, p = 0.003, η p 2 = 0.165), in which only DM females showed significantly lower freezing level compared with WT (p = 0.014) and Kmt2a-HET females (p = 0.003) ( Figure 3C). In females, neither the partial loss of Kdm5c nor Kmt2a showed impaired memory compared with WTs (both p = 1.0). There were no significant differences across genotypes in freezing or locomotor activity during training (both F(3,81) < 1) ( Figure 3A). Nevertheless, we did observe a significant effect of genotype in response to shock (F(3,81) = 7.46, p < 0.001. η p 2 = 0.225), driven by significantly higher locomotor activity burst in response to footshock in Kmt5c-HET females (cf WT: p = 0.02; cf Kmt2a: p < 0.001). DM females did not show this effect (cf WT: p = 0.31; cf Kmt2a: p = 0.12) ( Figure 3B).
This pattern of deficits in female DM, but not in Kdm5c-HET females, are somewhat surprising. We and others have observed impaired context fear conditioning in Kdm5c-KO males, and a rescue effect in double Kdm5c-KO, Kmt2a-HET males [13]. In females, we observed no significant deficits in Kdm5c-HETs, and substantially worse performance in DM mice in contextual fear conditioning. Importantly, the increased shock response in Kdm5c-HETs may indicate increased sensitivity of Kdm5c-HET females to shock that may compensate for a subtle memory impairment effect. Nevertheless, these results suggest a somewhat different interaction of histone methylation dynamics in memory processes in female animals compared to our previous male study.

Discussion
In this work, we characterize the transcriptome of female Kdm5c-HET mice in the developing brain and determine the effects of Kdm5c loss and Kmt2a-Kdm5c antagonisms on behavior in females. Our data indicate that the loss of KDM5C leads to both common and distinct gene expression changes between sexes, and these changes may underlie differential patterns across sex of behavioral traits in KDM5C disorder mouse models.
Commonly dysregulated genes in Kdm5c-deficient males and females may explain the behavioral traits seen in mice and human patients. Many MRXCSCJ patients have epilepsy [26], and Kdm5c-KO male mice have an increased propensity for kainic acid-induced seizures [31]. Our study highlights Gjb1, which encodes the protein connexin-32 that forms gap-junctions in oligodendrocytes and neurons [43], as a potential mediator of seizures ( Figure 1H). Most studies on Gjb1 focus on loss-of-function mutations, as they cause the sensorimotor neuropathy Charcot-Marie-Tooth type 1 [67]. Meanwhile, Gjb1 mRNA and connexin-32 protein levels increase when epileptic activity is induced by bicuculline in the hippocampus [44]. Such a Gjb1 overexpression may exacerbate seizure propensity with KDM5C deficiency. Two other characteristics of MRXSCJ and its mouse model are short stature and hyper aggression [22]. Cdkn1c, overexpressed in Kdm5c-deficient male and female developing forebrains, is a good candidate for potentially contributing to these two traits ( Figure 1I) for the following reasons. Reminiscent of patients and mice with KDM5C mutations, microduplications of the region harboring Cdkn1c cause Silver-Russell Syndrome, characterized by growth retardation and short stature [68,69]. Mice overexpressing Cdkn1c are small [70], and the male transgenic mice display hyper aggression in the social dominance tube test [46]. Currently, future studies are warranted to test the contribution of these candidate genes.
Genes whose expression changed uniquely in male or female Kdm5c-mutant mice illuminate the sexually-dimorphic manifestations of KDM5C deficiency. As female-specific DEGs, we highlighted Jpx and Mdk, both involved in early embryogenesis ( Figure 2). In particular, Jpx is a key player in X chromosome inactivation, a dosage compensation mechanism unique to female cells. KDM5C also regulates the expression of Xist, another critical lncRNA in X-inactivation in early embryogenesis [30]. The female-specific dysregulation of developmental genes such as Jpx and Xist may explain why Kdm5c-homozygous female embryos are lethal [30]. Meanwhile, some male-specific DEGs in Kdm5c mutants, such as Prmt and Glanin, have known roles in MRXSCJ-impaired behaviors, such as aggression and memory ( Figure 2C). Pnmt encodes an epinephrine-synthesis enzyme, and transgenic overexpression of this gene in mice results in elevated fighting behaviors in males but no changes in female behavior [48]. Galanin has an important role in learning and memory-galanin overexpression in the mouse forebrain impairs spatial learning [71][72][73], and this gene is overexpressed in the limbic system of post-mortem Alzheimer's disease patients [74]. These results imply that common traits between sexes, such as aggression and memory, may involve distinct mechanisms downstream of Kdm5c loss. However, our gene expression study was performed in P6 animals, and behavior was assessed in adults; thus, developmental contribution of gene misregulation awaits future validations. Regardless, the female-specific gene expression changes call attention to possible unique symptoms in MRXSCJ female patients.
This pattern of common and distinct dysregulation was also observed at the level of behavior. Whereas Kdm5c-HET females showed intact foreground fear conditioning, the double Kmt2a-Kdm5c knockout impaired memory performance. This pattern contrasts with the impaired memory in Kdm5c-KO and rescue in the double mutants we previously observed in male animals [13]. Female Kdm5c-HET mice-with and without Kmt2a mutation-also exhibited dysregulation of social behaviors. Kdm5c-HET mice showed social avoidance of a novel mouse instead of preference, and the Kmt2a-Kdm5c double mutants also failed to show social preference. Unlike in males, in which either Kmt2a or Kdm5c loss triggered hyperaggression [13], in females, neither Kmt2a-HET nor Kdm5c-HET alone altered social dominance. Nevertheless, consistent with that observed in males, the double mutant females showed a strikingly submissive phenotype.
The Kdm5c-HET females exhibited a milder hippocampal memory deficit compared with males, consistent with the weaker gene misregulation in female mice (Figure 1). Intriguingly, other studies have demonstrated that females do show impairments in the background-or cued-context fear conditioning tasks [31]. The foreground context fear conditioning protocol used here is less sensitive to disruption by hippocampal manipulations [75][76][77] than those that include a discrete cue, and females, in particular, may be more likely to recruit non-hippocampal circuits for context fear conditioning [38,77]. It is not that females are impervious to disruption, however. Unlike male Kdm5c-Kmt2a double mutants, which showed no fear memory deficits, female DMs were more impaired than their Kdm5c-HET counterparts.
Together, these findings demonstrate an overlapping but distinct pattern of gene misregulation and behavioral deficits in male Kdm5c-KO and female Kdm5c-HET mice that mimics the sex differences in symptomatology in individuals with MRXSCJ. Indeed, as with patients, some symptoms-including memory impairments and aggression-are less severe in female mice. These data suggest that other behaviors-and in particular social anxiety-may be more evident in girls with MRXSCJ. Importantly, these data also suggest that treatments targeting KDM5C or compensatory methylation enzymes may need to take sex into account carefully. In these studies, we have observed that whereas Kmt2a knockdown exerts a partial rescue for some behavioral deficits in males, the same genetic manipulation appears to unmask deficits in females. Although it remains likely that developmental effects play some role in the adverse effects of genetic intervention here, understanding sex differences in gene expression and behavioral symptomatology in MRXSCJ individuals will be essential for optimizing therapeutic interventions and strategies for both girls and boys.
Our work also provides insights into sex chromosome evolution. Kdm5c escapes X inactivation [39][40][41] (Figure 1). A unique feature of X-inactivation escapees is the presence of their paralogues in the Y chromosome [78,79]. KDM5D is the Y-linked paralogue for KDM5C in mice, humans, and other mammals [20]. Sex chromosomes are thought to originate from ordinary autosomes, and Y-chromosome genes have continued to be lost during the deterioration of Y. The persistence of the Y-linked paralogues of X-inactivation escapees led to the hypothesis that the X-Y pairs play the same roles that require a high dose achieved by expressing both X-and Y-genes, like many other autosomal genes [78]. This hypothesis predicts that Kdm5c loss in males will have the same impact as Kdm5c heterozygosity in females because KDM5C + KDM5D = 2x KDM5C. However, our data are discordant with this prediction. The unisexual DEGs showed more pronounced dysregulation in Kdm5c-KO males than in Kdm5c-HET females; KDM5D could not compensate for the loss of KDM5C ( Figure 1G). These data render the above hypothesis less tenable for this X-Y pair. Instead, our data suggest that KDM5C and KDM5D have distinct functions in behavior and brain gene expression.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12040637/s1, Table S1: all differentially expressed up-and downregulated genes found in males and females.